Anti-CD4 Anticorpo [GK1.5] (A86704)

Current influenza vaccines do not provide good protection against antigenically different influenza A viruses. As an approach to overcome strain specificity of protection, this study demonstrates significantly improved long-term cross protection by supplementing split vaccines with a conserved molecular target, a repeat of the influenza M2 ectodomain (M2e) expressed on virus-like particles (M2e5x VLPs) in a membrane-anchored form. Intramuscular immunization with H1N1 split vaccine (A/California/07/2009) supplemented with M2e5x VLPs induced M2e-specific humoral and cellular immune responses, and shaped the host responses to the vaccine in the direction of T-helper type 1 responses inducing dominant IgG2a isotype antibodies as well as interferon-γ (IFN-γ) producing cells in systemic and mucosal sites. Upon lethal challenge, M2e5x VLP-supplemented vaccination lowered lung viral loads and induced long-term cross protection against H3N2 or H5N1 subtype influenza viruses over 12 months. M2e antibodies, CD4 T cells, and CD8 T cells were found to contribute to improving heterosubtypic cross protection. In addition, improved cross protection by supplemented vaccination with M2e5x VLPs was mediated via Fc receptors. The results support evidence that supplementation with M2e5x VLPs is a promising approach for overcoming the limitation of strain-specific protection by current influenza vaccination.

Autologous tumor vaccine modified with nonlytic Newcastle disease virus (ATV-NDV) is a promising vaccine for cancer immunotherapy. IL-7 plays a critical role in lymphocyte development and homeostasis. To improve the efficacy of ATV-NDV, we inserted the murine IL-7 gene into the genome of nonlytic NDV strain LX using reverse genetic system. The insertion of the IL-7 gene neither affected the main features of NDV replication nor its tumor selectivity. The gene product was biologically active and stable. Then we tested the antitumor effects of the autologous tumor vaccine modified with LX/(IL-7) in the murine tumor models. We showed that tumor cells modified with LX/IL-7 induced a strong antitumor activity both in prophylaxis and therapeutic models. The IFN-γ production and the cytotoxicity of tumor-specific CD8(+) T cells were significantly enhanced after immunization with tumor cells modified with LX/(IL-7) in both models. Although the tumor-infiltrating CD4(+) T cells and CD8(+) T cells were both increased and their IFN-γ productions also were upregulated, the antitumor activity of the tumor vaccine modified with LX/(IL-7) was dependent on CD8(+) T cells. Our results demonstrated that the autologous tumor vaccine modified with NDV strain LX/(IL-7) could promote the antitumor immune responses mediated by CD8(+) T cells and significantly improve the efficacy of the ATV-NDV.

RH2 is a neurovirulent γ134.5 gene-deficient herpes simplex virus type 1 (HSV-1) with a lytic ability in human squamous cell carcinoma (SCC) cells; it is related to spontaneously occurring HSV-1 mutant HF10. The effect of RH2 on SCC was examined using a syngeneic C3H mouse model. After infection of mouse SCCVII cells with RH2, cell viability was decreased at first, but recovered by prolonged culture, indicating the limited replication of RH2. The antitumor ability of RH2 was examined using a bilateral SCCVII tumor model. The growth of the RH2-injected tumors was suppressed compared with that of phosphate-buffered saline-injected tumors. Moreover, the growth of contralateral tumor of RH2-treated mice was also suppressed significantly. The splenocytes of C3H mice treated with RH2 lysed more SCCVII cells than NFSaY83 cells and YAC-1 cells. The cytotoxicity of the splenocytes on SCCVII cells was significantly greater than that of splenocytes from tumor-bearing mice. Removal of CD8(+) T cells from splenocytes decreased their cell killing activity remarkably. The antitumor effect of RH2 on SCCVII xenografts in nude mice was not demonstrated. These results indicate that RH2 exhibited a suppressive effect on mouse SCC, even if the replication of RH2 was limited. This is ascribed to the ability of RH2 to enhance existing tumor-specific cytotoxic T lymphocyte activity.

Monocyte infiltration and subsequent differentiation into macrophages has been shown to be crucial during inflammation. Metalloproteinases are key enzymes in these processes, but the role of MMP-14 remains largely unknown. To address this question, we generated animals with conditional ablation of MMP-14 in the monocyte/macrophage lineage. The knockout (KO) animals (LysM-Cre(+)MMP-14(fl/fl)) were healthy and fertile, and neither skin architecture nor differentiation was altered from the wild type (WT). Full-thickness wounds were induced, and careful analysis of wound closure, granulation tissue formation, and angiogenesis revealed no differences between genotypes. The inflammatory response, monocyte influx, differentiation, and lymphocyte infiltration was also similar in KO and WT animals. Ear swelling after croton oil application was similar in the KO and WT animals. Interestingly, the number of monocytes and macrophages, as well as of T cells, was significantly reduced in KO animals, compared with WT animals. Similarly, both P-selectin and proinflammatory cytokine levels were markedly reduced in KO animals. In vitro, the migratory capacity of isolated KO macrophages was significantly impaired on fibronectin, a substrate of MMP-14. These data point to a role of MMP-14 during transendothelial migration of monocytes and T-cell attraction.

The chromosomal translocation t(4;11)(q21;q23) is the most frequent genetic aberration of the human MLL gene, resulting in high-risk acute lymphoblastic leukemia (ALL). To elucidate the leukemogenic potential of the fusion proteins MLL.AF4 and AF4.MLL, Lin(-)/Sca1(+) purified cells (LSPCs) were retrovirally transduced with either both fusion genes or with MLL.AF4 or AF4.MLL alone. Recipients of AF4.MLL- or double-transduced LSPCs developed pro-B ALL, B/T biphenotypic acute leukemia, or mixed lineage leukemia. Transplantation of MLL.AF4- or mock-transduced LSPCs did not result in disease development during an observation period of 13 months. These findings indicate that the expression of the AF4.MLL fusion protein is capable of inducing acute lymphoblastic leukemia even in the absence of the MLL.AF4 fusion protein. In view of recent findings, these results may imply that t(4;11) leukemia is based on 2 oncoproteins, providing an explanation for the very early onset of disease in humans.

Monoclonal antibody GK1.5 recognizes a previously undescribed murine T cell surface molecule, designated L3T4, which migrates on SDS-PAGE under reducing conditions as a single band with an apparent m.w. of 52,000. L3T4 is expressed by approximately 80% of thymocytes and by approximately 20% of spleen cells. There appears to be poor correlation between expression of L3T4 by functional T cell clones and expression of Lyt-2, expression of the cytolytic phenotype, and class I MHC antigen reactivity. On the other hand, both a class II MHC antigen-reactive HTL clone and an Lyt-1- Mls-reactive HTL clone express L3T4. Analysis of the effect of mAb GK1.5 on PFC responses in adoptive transfer suggests that L3T4 is expressed by the helper/inducer subset of murine T cells. Expression of L3T4 by murine T cells, however, may correlate primarily with class II MHC antigen reactivity rather than with functional phenotype; mAb GK1.5 profoundly blocks antigen-specific cytolysis by the cloned class II MHC antigen-reactive CTL line A15-1.17. Antigen-specific cytolysis by A15-1.17 is blocked by mAb GK1.5 at a step before the lethal hit. Collectively, the flow cytometric, functional, and biochemical data indicate that L3T4 is similar to the human Leu-3/T4 molecule.

BACKGROUND:

Glioblastoma (GBM) is the deadliest of brain tumors. Standard treatment for GBM is surgery, followed by combined radiation therapy and chemotherapy. Current therapy prolongs survival but does not offer a cure. We report on a novel immunotherapy against GBM, tested in an animal model of C57BL/6 mice injected intra-cranially with a lethal dose of murine GL261 glioma cells.

METHODS:

Ten week-old C57BL/6 mice were anesthetized before injection of 2 × 10(4) GL261 cells in the right cerebral hemisphere and after 3 days half of the mice were administered a single subcutaneous (s.c.) injection of irradiated semi-allogeneic vaccine, while mock-vaccinated mice received a s.c. injection of phosphate-buffered saline (PBS). Tumor engraftment was monitored through bioluminescence imaging (BLI). Length of animal survival was measured by Kaplan-Meier graphs and statistics. At time of sacrifice brain tissue was processed for estimation of tumor size and immunohistochemical studies.

RESULTS:

Overall survival of vaccinated mice was significantly longer compared to mock-vaccinated mice. Five to ten percent of vaccinated mice survived more than 90 days following the engraftment of GL261 cells in the brain and appeared to be free of disease by BLI. Tumor volume in the brain of vaccinated mice was on average five to ten-fold smaller compared to mock-vaccinated mice. In vaccinated mice, conspicuous microglia infiltrates were observed in tumor tissue sections and activated microglia appeared to form a fence along the perimeter of the tumor cells. The results of these animal studies persuaded the Office of Orphan Products Development of the Food and Drug Administration (FDA) to grant Orphan Drug Designation for treatment of GBM with irradiated, semi-allogeneic vaccines.

CONCLUSIONS:

Our preclinical observations suggest that semi-allogeneic vaccines could be tested clinically on subjects with GBM, as an adjuvant to standard treatment.

OBJECTIVE:

T helper 17 cells and interleukin-17A have been implicated in the progression of abdominal aortic aneurysm (AAA). Retinoic acid-related orphan receptor gamma thymus, the master transcription factor of T helper 17 cell differentiation, is selectively antagonized by digoxin. However, the effect of antagonizing retinoic acid-related orphan receptor gamma thymus on AAA has not been investigated.

APPROACH AND RESULTS:

We used human aortic sample analysis and 2 different experimental AAA models: (a) Angiotensin II (Ang II)-induced ApoE(-/-) male mice (Ang II/APOE model) and (b) porcine pancreatic elastase perfusion C57BL/6 mice (porcine pancreatic elastase/C57 model). In the Ang II/APOE model, all mice (n=80) were divided into 4 groups: sham group (saline+0.5% dimethyl sulfoxide treatment), control group (Ang II+0.5% dimethyl sulfoxide treatment), low-dose group (Ang II+low-dose digoxin, 20 μg/d per mouse), and high-dose group (Ang II+high-dose digoxin, 40 μg/d per mouse). All treatments began on day 0 after surgery. Efficacy was determined via aortic diameter and systolic blood pressure measurements, histopathology and protein expression, and flow cytometry analysis when euthenized. Human aortic tissue analysis showed that both interleukin-17A and retinoic acid-related orphan receptor gamma thymus increased in AAA tissues. The low-dose and high-dose groups had AAA incidences of 60% and 35%, respectively, compared with 70% in the control group. The T helper 17- and interleukin-17A-related inflammatory responses were dose-dependently attenuated by digoxin treatment. Digoxin was also highly effective in the porcine pancreatic elastase/C57 model.

CONCLUSIONS:

Digoxin attenuates experimental AAA progression in a model-independent manner. Antagonizing retinoic acid-related orphan receptor gamma thymus activity by digoxin may become a novel strategy for nonsurgical AAA treatment.

© 2014 American Heart Association, Inc.

PURPOSE:

To assess the frequency and the functional characteristics of one major component of immune tolerance, the CD4(+)FoxP3(+) regulatory T cells (Tregs) in a mouse model of abdominal irradiation.

METHODS AND MATERIALS:

Mice were exposed to a single abdominal dose of γ-radiation (10 Gy). We evaluated small intestine Treg infiltration by Foxp3 immunostaining and the functional suppressive activity of Tregs isolated from mesenteric lymph nodes.

RESULTS:

Foxp3 immunostaining showed that radiation induced a long-term infiltration of the intestine by Tregs (levels 5.5 times greater than in controls). Co-culture of Tregs from mesenteric lymph nodes with CD4(+) effector cells showed that the Tregs had lost their suppressive function. This loss was associated with a significant decrease in the levels of Foxp3, TGF-β, and CTLA-4 mRNA, all required for optimal Treg function. At Day 90 after irradiation, Tregs regained their suppressive activity as forkhead box P3 (Foxp3), transforming growth factor beta (TGF-β), and cytotoxic T-lymphocyte antigen 4 (CTLA-4) expression returned to normal. Analysis of the secretory function of mesenteric lymph node Tregs, activated in vitro with anti-CD3/anti-CD28 Abs, showed that this dysfunction was independent of a defect in interleukin-10 secretion.

CONCLUSION:

Radiation caused a long-term accumulation of function-impaired Foxp3(+)CD4(+) Tregs in the intestine. Our study provides new insights into how radiation affects the immune tolerance in peripheral tissues.

Copyright © 2011 Elsevier Inc. All rights reserved.